Detection of Oral Human Papillomavirus (HPV) and its Clinical Importance

Statement of the Problem: Human papillomavirus (HPV) has a tropism for the squamous epithelium and cause a wide range of diseases, from benign lesions to invasive tumors that can affect the oral cavity. Purpose: This study aimed to estimate HPV infection in compatible stomatological lesions. Materials and Method: A cross-sectional study was carried out from March 2017 to August 2019, which included patients who attended the Oral Medicine Department of the School of Dentistry of the University of Buenos Aires who presented oral manifestations compatible with HPV infection that accepted to be studied by histopathology and determination of viral genotype by polymerase chain reaction (PCR). The study was carried out from the biopsy fixed in formalin and included in paraffin, for histopathological study and the genotypification of HPV by genotype-specific PCR and/or sequencing of the L1 fragment. To confirm the negative cases hybrid capture method was also used. The 95% OR-IC was estimated. Results: 108 patients, 76 women and 32 men were studied, who underwent a clinical stomatological examination and genotyping of HPV (PCR-specific genotype), being positive for 60 patients and negative for 48. Among the positive cases (n= 60) 46.7% (n= 28) corresponded to elevated lesions infected with high-risk HPV genotypes, 43.3% (n= 26) to elevated lesions with low-risk HPV genotypes, regarding flat lesions it was found that 5% (n=3) corresponded with high-risk HPV genotypes and another 5% (n=3) with low-risk genotypes, with OR 1,076 95% CI (0.1993-5.818). The HPV genotypes found were 2, 6, 11, 13, 16, 18, 26, 31, 32, 33, 35, 51, 58, 64 and 72. Conclusion: Our results estimated an association between white, bright, and elevated oral lesions and the presence of high-risk HPV.

Introduction environment for initial HPV infection and its subsequent persistence, increasing the risk of transmission and its carcinogenic potential [6]. HPV is spread by direct cell-to-cell contact without the classic signs of viremia [7]. After entering the cell, the viral genome is taken to the cell nucleus where it is translated and transcribed. The replication of the viral genome follows some stages; at first stage, E1 and E2 early proteins are synthesized. As a result, 10 (9). Oral HPV infections have been related to sexual behavior, but recent evidence supports their horizontal mouth-tomouth transmission. Most HPV infections in babies are acquired vertically from the mother during the intrauterine period, during childbirth, or later through saliva [2].

Materials and Method
This study was part of the "Rodolfo Erausquin" Clinical

Data collection
All the data was collected in a protocolled medical history that included age, sex, clinical characteristics of the lesion, and its location. Laboratory tests (blood count, coagulogram, liver function test, glycemia, uremia, uricemia, erythrocyte sedimentation) were requested from all patients to assess their general condition.

Assessment of oral lesions
The evaluation of the stomatological clinical lesions was carried out by stomatologists and the OPMI PICO S100 CARL ZEISS clinical operating microscope was  Figure 1d). Regarding the appearance of the lesions, we observed that they could be shiny or translucent (these are white lesions with leukoedema, when dried they do not modify their aspect, they remain bright) and opaque.

Sample Collection
Biopsies were performed in the affected areas and divided into two parts, one was fixed in 10% formalin, included in paraffin, and stained with hematoxylin-eosin for its pathological study and the other was stored in saline solution for its determination of HPV, which was analyzed by nested PCR and subsequent sequencing using MY09 /11 and GP05+/06+ primers. It was incubated with 200ul of lysis buffer (Lysis tissue buffer, Roche) and 20ul of proteinase K with shaking at 650 RPM in a thermostatic bath for 12 hours [10].

Results
All patients with a diagnosis of high-risk HPV continue to be followed up to this day.

Discussion
A stomatologist makes the first clinical presumption of the presence of HPV in the oral mucosa through the ide-  No other authors found in the literature took these oral lesions as a reference as suspects of HPV infection.
Like Ribeiro M et al. [18], to improve the detection of HPV, we used the nested PCR technique that uses more than one pair of primers. Therefore, we were able to detect the virus even at very low concentrations. Highquality DNA is required for this technique to reach its optimal conditions [18]. PCR amplification of HPV DNA is a target amplification technique that is capable of amplifying traces of DNA sequences in a biological sample containing heterogeneous cell types. This technique is very sensitive but also it has low specificity and clinical samples are very prone to cross-contamination.
In this study, we found the presence of oral infection by multi-type HPV (2 types) in 7% of the patients (n= 4) and most of them presented high-risk HPV genotypes. Bui et al. [22] reported that the prevalence of multiple oral HPV infection (2 to 6 types) was 1.5% (2.5% for men, 0.4% for women). The majority of oral multitype HPV cases (83.8%) harbored one or more oncogenic types [22]. In this study, the prevalence of HPV was 56% and 13% corresponded to HPV 16. Gillison et al. [23] state that the prevalence of oral HPV infection among men and women between the ages of 14 and 69 in the United States was 6.9% and HPV type 16 was 1% [23]. Sonawane et al. [24] postulated that the general prevalence of oral HPV infection was 11.5%.
Many studies are addressing the presence of HPV in the oral cavity of healthy, uninjured patients. Our study considered the clinical stomatological lesions compatible with HPV infection. This is why the prevalence of this study was higher than that of the studies cited above.

Conclusion
PCR is a very important technique to determine the HPV genotype present in these lesions due to its high sensitivity. We found an association between high-risk HPV and elevated white lesions, these oncogenic HPVs being a risk factor for the development of these lesions.
Further studies need to be made to assess the clinical and molecular implications of the virus in other lesions and the carcinogenic process.